Major trauma can cause volumetric muscle loss (VML) resulting in life-long disability. Current therapies are extremely limited and offer little hope of meaningful functional recovery. We have pursued a Regenerative Rehabilitation approach to VML therapy by combining stem cell transplantation technology with physical activity in the form of voluntary wheel running to promote the engraftment and maturation of the transplanted cells. In a murine model of VML, we have been developing methodologies for the transplantation of muscle stem cells (MuSCs) along with other mononucleated cells isolated from muscle by FACS into VML lesions. Based on both histological and physiological assessment, we are able to obtain significant recovery of muscle structure and function. However, even though there is significant recovery of force as assessed by direct electrical stimulation of the muscle ex vivo, force assessment by stimulation of the sciatic nerve in vivo has revealed minimal recovery, suggesting that innervation of the newly formed muscle is limited. Likewise, the enhancement of regenerative responses by physical activity, such as enhanced vascularity and reduced fibrosis, are not accompanied by significant increases in muscle fiber cross-sectional area, suggesting that this form of exercise does yield a significantly greater amount of axon regrowth and neuromuscular junction (NMJ) formation and maturation. Based on evidence of effects of different forms of exercise on axon regrowth, the focus of the studies of this proposal is to examine different exercise paradigms to optimize MuSC therapy for VML by enhancing axon regrowth and NMJ formation. The studies are divided into two Specific Aims. In Aim 1, we will initially compare two types of treadmill training (low intensity training and high intensity interval training) to voluntary wheel running i simple models of re- innervation - VML injury alone (i.e. no transplantation) and a combined muscle/nerve injury model (injection of the myotoxin cardiotoxin and the neurotoxin batrachotoxin). The former provides evidence as to early responses seen after VML plus transplantation, and the latter provides a model of re-innervation in a setting of extensive muscle regeneration. We will assess NMJ formation histologically and force generation from sciatic nerve stimulation. In Aim 2, we will apply whichever exercise paradigm leads to the best outcome to the study of effects of exercise on MuSC transplantation for VML. We will use our optimized transplantation protocol following VML surgery and then apply the optimized exercise protocol for three weeks. At that time, we will again assess the restoration of muscle structure and function by histological and physiological analyses. The long-term goal is to optimize Regenerative Rehabilitation strategies for the treatment of VML in humans that involve a combination of regenerative medicine approaches, in the form of stem cell therapeutics, with rehabilitation medicine approaches, in the form of physical activity, to optimize tissue restoratio and functional recovery. As such, all of the approaches are designed to be scalable to humans and to model as closely as possible the challenges faced by humans with VML.